Pic1 inhibition of myeloperoxidase oxidative activity in an animal model

ABSTRACT

A method of treating systemic lupus erythematosus in a subject is provided in which a therapeutically effective amount of PIC1 is administered to the subject. A method of treating transfusion-related acute lung injury is also provided where a therapeutically effective amount of PIC1 is administered to the subject. PIC1 can modulate immune complex activation of the complement system and NET formation in the subject. PIC1 can also inhibit myeloperoxidase (MPO) activity in the subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/615,183, filed Jan. 9, 2018, U.S. Provisional Application No.62/681,458, filed Jun. 6, 2018, and U.S. Provisional Application No.62/746,649, filed Oct. 17, 2018, the disclosures of each which areherein incorporated by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 7, 2019, isnamed Seqlist.txt and is 16,143 bytes in size.

BACKGROUND

NETs are a means by which neutrophils contain infection by turningthemselves into webs of DNA decorated with antimicrobial molecules. NETshave been shown to contribute to pathogenesis in a wide range ofinflammatory and autoimmune diseases, such as Systemic LupusErythematosus (SLE) and transfusion-related acute lung injury (TRALI).

The pathogenesis of SLE is very complex, but two major contributors areimmune complex-initiated complement activation and NET formation. Immunecomplexes that initiate classical pathway complement activation leadingto consumption of C4 and C3 have long been appreciated and contribute toLupus Nephritis [1-3]. NETs, however, are more recently recognized ascontributing to SLE pathogenesis [4-6].

TRALI is the leading cause of morbidity and mortality associated withblood transfusion. TRALI is defined as an acute lung injury that occurswithin 6 hours of receiving an allogenic blood product transfusion.TRALI is commonly characterized by dyspnea, fever, hypotension,hypoxemia and pulmonary edema with laboratory tests demonstratingtransient leukopenia and thrombocytopenia and bilateral infiltrates bychest X-ray. The mortality rate is 5-10% with a majority of patients(70-90%) requiring mechanical ventilation and hemodynamic support. Inthe absence of efficacious pharmacological intervention, the currentstandard of care is limited solely to supportive therapy.

The pathophysiology of TRALI is complex. Clinically, antibodies to humanleukocyte antigens or human neutrophil alloantigens in donor bloodproducts are believed to cause the majority of TRALI cases. Non-antibodymediated or non-immune TRALI, usually resulting after platelet orerythrocyte transfusion, accounts for 11-39% of cases. A number ofanimal models have been established to investigate the pathophysiologyof both antibody-mediated and non-antibody mediated TRALI. Whileneutrophils play a key part in the pathogenesis of antibody-mediatedTRALI, clinical reports and animal model data suggest that depending onthe class of antibodies involved, monocytes, lymphocytes, platelets aswell as endothelial cells may contribute to TRALI. Complement activationis also required for the development of TRALI in variousantibody-mediated animal models, and contributes to the pathologicalprocess in clinical TRALI cases.

Animal models have demonstrated that antibody-mediated activation ofhost neutrophils induces sequestration in the pulmonary capillaries thatleads to tissue injury. These activated neutrophils release neutrophilextracellular traps (NETs) which contribute to TRALI pathogenesis inmouse models. NET biomarkers, e.g., myeloperoxidase (MPO), nucleosomesand DNA, have been detected in serum collected from TRALI patients.

Myeloperoxidase (MPO) is a heme-based peroxidase found in neutrophils.MPO is the major enzyme present in neutrophils, accounting for 30% ofthe dry weight of the cell. The main function of MPO is to generatehypochlorous acid to help neutrophils kill microbial invaders. However,generation of hypochlorous acid by MPO can also lead to the damage ofhost tissues and has been shown to directly contribute to parenchymalinjury in many inflammatory diseases. For instance, MPO contributes toparenchymal lung damage in cystic fibrosis. In addition to directantimicrobial activity, MPO has also been shown to act in the importantpathway of creating neutrophil extracellular traps (NETs).

BRIEF DESCRIPTION OF THE DRAWINGS

This patent application file contains at least one drawing executed incolor. Copies of this patent application with color drawing(s) will beprovided by the Office upon request and payment of the necessary fee.

FIGS. 1A-1C are graphs showing PA-dPEG24 inhibition of heat-aggregatedIgG immune complex-initiated complement activation assayed by complementeffectors. FIG. 1A shows PA-dPEG24 inhibited C5a generation in normalhuman serum (NHS) stimulated with heat-aggregated IgG (Agg-IgG) immunecomplexes. The data shown are means of (n=5) independent experiments andthe standard error of the mean (SEM). FIG. 1B shows PA-dPEG24 inhibitionof iC3b generation in normal human serum (NHS) stimulated withheat-aggregated IgG (Agg-IgG) immune complexes. The data shown are meansof (n=4) independent experiments and the standard error of the mean(SEM). FIG. 1C shows PA-dPEG24 inhibition of SC5b-9 generation in normalhuman serum (NHS) stimulated with heat-aggregated IgG (Agg-IgG) immunecomplexes. The data shown are means of (n=6) independent experiments andthe standard error of the mean (SEM).

FIGS. 2A-2C are graphs showing PA-dPEG24 inhibition ofovalbumin-antiovalbumin immune complex-initiated complement activationassayed by complement effectors. FIG. 2A shows PA-dPEG24 inhibition ofC5a generation in normal human serum (NHS) stimulated withovalbumin-antiovalbumin immune complexes. The data shown are means of(n=4) independent experiments and the standard error of the mean (SEM).FIG. 2B shows PA-dPEG24 inhibition of iC3b generation in normal humanserum (NHS) stimulated with ovalbumin-antiovalbumin immune complexes.The data shown are means of (n=4) independent experiments and thestandard error of the mean (SEM). FIG. 2C shows PA-dPEG24 inhibition ofSC5b-9 generation in normal human serum (NHS) stimulated withovalbumin-antiovalbumin immune complexes. At 2 mM PA-dPEG24 the measuredSC5b-9 was at the lower limit of detection. The data shown are means of(n=3) independent experiments and the standard error of the mean (SEM).

FIGS. 3A-3C are graphs showing PA-dPEG24 inhibition of C1-antiC1q immunecomplex-initiated complement activation assayed by complement effectors.FIG. 3A shows PA-dPEG24 inhibition of C5a generation in normal humanserum (NHS) stimulated with C1-antiC1q immune complexes. The data shownare means of (n=4) independent experiments and the standard error of themean (SEM). FIG. 3B shows PA-dPEG24 inhibition of iC3b generation innormal human serum (NHS) stimulated with C1-antiC1q immune complexes.The data shown are means of (n=4) independent experiments±SEM. FIG. 3Cshows PA-dPEG24 inhibition of SC5b-9 generation in normal human serum(NHS) stimulated with C1-antiC1q immune complexes. The data shown aremeans of (n=4) independent experiments and the standard error of themean (SEM).

FIG. 4 shows PA-dPEG24 inhibition of PMA-initiated NET formation withhuman neutrophils (PMN) assayed by fluorescence microscopy and PicoGreenquantitation of free DNA. The first row shows unstimulated neutrophils,the second row shows neutrophils stimulated with PMA and hydrogenperoxide (H₂O₂) and the third row shows neutrophils stimulated withPMA+H₂O₂ in the presence of 5 mM PA-dPEG24 (PIC1). The first columnshows slides probed with DAPI to visualize DNA and the second columnshows slides probed with anti-MPO antibody. The graph shows PA-dPEG24 (5mM) inhibition of NET generation by human neutrophils stimulated withPMA+H₂O₂ assayed by PicoGreen. The data shown are means of (n=3)independent experiments and the standard error of the mean (SEM).

FIG. 5 shows PA-dPEG24 inhibition of PMA-initiated NET formation withhuman neutrophils (PMN) assayed by fluorescence microscopy for DNA(DAPI), neutrophil elastase (anti-NE antibody), and histone H3(anti-histone H3 antibody). The first row shows unstimulatedneutrophils, the second row shows neutrophils stimulated with PMA andhydrogen peroxide (H₂O₂) and third row shows neutrophils stimulated withPMA+H₂O₂ in the presence of 5 mM PA-dPEG24 (PIC1). The first columnshows slides probed with DAPI to visualize DNA, the second column showsslides probed with anti-neutrophil elastase antibody and the third rowshows slides probed with anti-histone H3 antibody. Representative imagesare shown.

FIG. 6 shows PA-dPEG24 inhibition of MPO-initiated NET formation withhuman neutrophils (PMN) assayed by fluorescence microscopy and PicoGreenquantitation of free DNA. The first row shows unstimulated neutrophils,the second row shows neutrophils stimulated with MPO and hydrogenperoxide (H₂O₂), the third row shows neutrophils stimulated withMPO+H₂O₂ in the presence of PA-dPEG24 (PIC1) and the fourth row showsneutrophils incubated with PA-dPEG24 (PIC1) only. The first column showsslides probed with DAPI to visualize DNA. The second column shows slidesprobed with anti-MPO antibody. The graph shows PA-dPEG24 inhibition ofNET generation by human neutrophils stimulated with MPO+hydrogenperoxide assayed by PicoGreen. The data shown are means of (n=3)independent experiments and the standard error of the mean (SEM).

FIGS. 7A-7C show PA-dPEG24 inhibition of immune complex-activatedserum-initiated NET formation with human neutrophils (PMN) assayed byfluorescence microscopy and PicoGreen quantitation of free DNA. FIG. 7Ashows NET formation induced by PMNs incubated alone (untreated), withnormal human sera (NHS), immune complexes alone (IC) or immunecomplex-activated sera (ICsera). FIG. 7B shows PA-dPEG24 inhibition ofNET generation by human neutrophils stimulated with immunecomplex-activated sera (ICsera) assayed by PicoGreen. The data shown aremeans of (n=4) independent experiments and the standard error of themean (SEM). In FIG. 7C, the first row shows unstimulated neutrophils,the second row shows neutrophils stimulated with immunecomplex-activated human sera (ICsera) and hydrogen peroxide (H₂O₂) andthe third row shows neutrophils stimulated with ICsera+H₂O₂ in thepresence of PA-dPEG24 (PIC1). The first column shows slides probed withDAPI to visualize DNA and the second column shows slides probed withanti-MPO antibody.

FIGS. 8A and 8B show the results of optimization of human RBCtransfusion into rats. In FIG. 8A, free hemoglobin present in rat plasmacollected before transfusion (0) or 0.5, 5, 20, 60, 120 or 360 min after15 (♦, n=3), 30 (▴, n=3) or 45% (▪, n=3) transfusion of human RBCs wasmeasured by spectrophotometry. One group of sham animals (●, n=3) wasanalyzed as well. In FIG. 8B, the percent of surviving human RBCs from15 (●, n=3), 30 (▪, n=3) or 45% (▴, n=3) transfusion of human RBCs weredetected using FITC-conjugated anti-human CD235a (glycophorin A)monoclonal antibody at 0.5, 5, 20 60, 120 and 360 min after transfusionas measured by flow cytometry. Clearance kinetics were standardized toinjected RBCs at baseline (0 min). The data shown are means and standarderror of the mean (SEM).

FIGS. 9A-9D show that an LPS “first-hit” is required for transfusioninduced lung injury. Representative histology (hematoxylin and eosin)stains are shown of lungs from sham rats (FIG. 9A), rats receiving 30%(FIG. 9B), 45% transfusion of mismatched RBCs in the absence of LPS(FIG. 9C) and rats receiving 30% transfusion after LPS administration(FIG. 9D). Animals receiving transfusion in the absence of LPSdemonstrated normal lung architecture as seen in sham treated animalswhereas animals receiving LPS prior to 30% transfusion showed severeneutrophil infiltration and thickening of the alveolar cell walls. Barrepresents 100 μm. Tissues were observed with a microscope (BX50,Olympus) at a magnification of 20× at room temperature. Images wereacquired with a digital camera (DP70, Olympus).

FIGS. 10A-10C are graphs showing that an LPS “first-hit” inducesleukopenia. Blood was collected from rats before infusion of 30%mismatched RBC not receiving LPS pre-treatment or with LPS pre-treatment(pre-transfusion, n=22). Four hours after transfusion, blood was againcollected from rats transfused without (transfusion only, n=5) or withLPS (transfusion+LPS, n=3). Levels of WBCs and neutrophils (FIG. 10A),monocytes (FIG. 10B) and lymphocytes (FIG. 10C) are reported. The datashown are means and standard deviation of the mean.

FIGS. 11A-11D show that prophylactically administered PIC1 attenuatesacute lung injury. FIG. 11A shows gross lung weights measured for shamanimals (n=7), animals treated prophylactically with vehicle (n=5) orPIC1 (n=8). The data shown are means and standard error of the mean(SEM). Representative histology (hematoxylin and eosin) stains are shownof lungs from sham rats (FIG. 11B), rats receiving vehicle (FIG. 11C)and rats receiving PIC1 (FIG. 11D). Animals receiving PIC1 demonstratedthe normal lung architecture as seen in sham treated animals, whileanimals receiving vehicle showed consolidation of the alveolar spacesand thickening of the alveolar cell walls. The bar represents 100 μm.Tissues were observed with a microscope (BX50, Olympus) at amagnification of 20× at room temperature. Images were acquired with adigital camera (DP70, Olympus).

FIGS. 12A-12B are graphs showing that PIC1 reduces neutrophil-mediatedlung injury and MPO activity in the lungs. In FIG. 12A, blinded gradingof H&E sections for neutrophil infiltration and cell wall thickeningform animals receiving vehicle (n=7) and animals receiving PIC1 (n=9)were scored on a scale of 0-4: 0=normal lungs, 1=minor lung involvement,2=moderate lung involvement, 3=serious lung involvement, 4=severe lunginvolvement. The box shows quartiles, the whiskers are 25th percentile,and the solid line is the mean. In FIG. 12B, MPO was isolated fromhomogenized lung tissue by antibody capture to measure MPO activity.Samples were combined with hydrogen peroxide and ADHP solution andimmediately read at an excitation wavelength of 535 nm and emissionwavelength of 590 nm in a microplate reader from 0 to 10 minutes every25 seconds. MPO (positive control, +CTR, n=3) and PBS (negative control,−CTR, n=3) were analyzed along with samples from animals receivingvehicle (n=2) and animals receiving PIC1 (n=3). Each sample wasevaluated in triplicate. The data shown are means and the standarddeviation of the mean. For simplicity, only the 5- and 10-minute timepoints are shown.

FIGS. 13A-13D are graphs showing that PIC1 reduces leukopenia. Blood wascollected from rats receiving vehicle (n=5) or PIC1 (n=8). Levels ofWBCs (FIG. 13A), lymphocytes (FIG. 13B), neutrophils (FIG. 13C) andmonocytes (FIG. 13D) are reported. The data shown are means and standarderror of the mean (SEM).

FIG. 14 is a graph showing that PIC1 reduces the level of free DNA incirculation. Plasma samples from animals receiving vehicle (n=3) or PIC1(n=3) were incubated with PicoGreen. Fluorescence was read at anexcitation wavelength of 485 nm and an emission wavelength of 520 nm ina microplate reader. All free DNA measurements for each animal were donein triplicate. The data shown are means and standard error of the mean(SEM).

FIG. 15 illustrates a model of PIC1 inhibition of TRALI. In this animalmodel, the first hit of LPS followed by 30% human RBC transfusionresults in a TRALI-like phenotype consisting of lung damage mediated byneutrophil sequestration and activation leading to MPO-mediated reactiveoxygen species (ROS) generation and NETosis. Free heme from thecomplement-mediated hemolysis also contributes to ROS formation. PIC1can inhibit complement-mediated hemolysis, C3a and C5a generation, ROSformation as well as MPO-mediated NETosis and ROS formation thusinhibiting TRALI. The complement anaphylatoxins C3a and C5a are show asstippled arrows as their direct role in neutrophil activation in thismodel is unknown.

FIG. 16 is a graph showing PIC1 dose-response inhibition of MPO-mediatedoxidation of TMB for increasing concentrations of MPO.

FIG. 17 is a graph in which increasing doses of purified MPO injected byIP show increased TMB peroxidation in peritoneal wash samples.Increasing amounts of purified MPO were injected IP and after one hour,animals underwent phlebotomy, euthanasia and peritoneal wash. Peritonealwash supernatant oxidation of TMB (n=4) was measured. The data shown aremeans of independent animals±SEM.

FIG. 18 is a graph in which increasing doses of purified MPO IPdemonstrate increased free DNA in peritoneal wash samples. Increasingamounts of purified MPO was injected IP and after one hour, animalsunderwent phlebotomy, euthanasia and peritoneal wash. Peritoneal washsupernatant free DNA was measured via PicoGreen assay (n=4). The datashown are means of independent animals±SEM.

FIG. 19 is a graph showing the results of intraperitoneal MPO timecourse experiments. Purified MPO (0.1 mg) was injected IP and atincreasing intervals animals underwent phlebotomy, euthanasia andperitoneal wash. Peritoneal wash supernatant oxidation of TMB (n=4) wasmeasured. The data shown are means of independent animals±SEM.

FIG. 20 is a graph showing the results of intraperitoneal MPO timecourse experiments. Purified MPO (0.1 mg) was injected IP and atincreasing intervals animals underwent phlebotomy, euthanasia andperitoneal wash. Peritoneal wash supernatant free DNA was measured viaPicoGreen assay (n=4). The data shown are means of independentanimals±SEM.

FIGS. 21-23 are graphs showing intraperitoneal MPO with increasing dosesof PIC1. Purified MPO (0.1 mg) was injected IP immediately followed byPIC1 injection IP at increasing doses. Two (2) hours after IPinjections, animals underwent phlebotomy, euthanasia and peritonealwash. FIG. 21 shows peritoneal wash supernatant oxidation of TMB (n=4).FIG. 22 shows peritoneal wash supernatant free DNA measured viaPicoGreen assay (n=4). FIG. 23 shows blood plasma free DNA measured viaPicoGreen assay (n=4). In FIGS. 21-23, the data shown are means ofindependent animals±SEM.

SUMMARY OF THE INVENTION

In one aspect is provided a method of treating systemic lupuserythematosus (SLE) in a subject. The method comprises administering atherapeutically effective amount of PIC1 to the subject.

In some embodiments, the method is effective to modulate immune complexactivation of the complement system and NET formation in the subject. Insome embodiments, the NET formation is stimulated by at least one of abacterium, a fungus, a parasite or a virus. In some embodiments, themethod is effective to inhibit NET-mediated inflammatory tissue damagein the subject.

In another aspect is provided a method of treating transfusion-relatedacute lung injury (TRALI) in a subject. The method comprisesadministering a therapeutically effective amount of PIC1 to the subject.

In some embodiments, the method is effective to modulate immune complexactivation of the complement system and NET formation in the subject. Insome embodiments, the NET formation is stimulated by at least one of abacterium, a fungus, a parasite or a virus. In some embodiments, themethod is effective to inhibit NET-mediated inflammatory tissue damagein the subject. In some embodiments, the PIC1 is administered before thesubject is administered a blood transfusion, after the subject isadministered the blood transfusion, and/or during the blood transfusion.

In various embodiments of the above aspects, the PIC1 inhibitsmyeloperoxidase (MPO) activity in the subject. In various embodiments,the PIC1 is administered parenterally. In various embodiments, thesubject is human. In certain embodiments, the PIC1 is a peptidecomprising one or more PEG moieties. In certain embodiments, the PIC1 isPA-dPEG24. In certain embodiments, the PA-dPEG24 comprises the sequenceof IALILEPICCQERAA-dPEG24 (SEQ ID NO: 19).

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, suitable methods and materials are described below.

The term “inhibition” refers to the reduction in the biological functionof an enzyme, protein, peptide, factor, byproduct, or derivative thereofeither individually or in complexes; reduction in the quantity of abiological protein, peptide, or derivative thereof whether in vivo or invitro; or interruption of a biological chain of events, cascade, orpathway known to comprise a related series of biological or chemicalreactions. The term “inhibition” may thus be used, for example, todescribe the reduction of quantity of a single component of thecomplement cascade compared to a control sample, a reduction in the rateor total amount of formation of a component or complex of components, orthe reduction of the overall activity of a complex process or series ofbiological reactions leading to such outcomes as cell lysis, formationof convertase enzymes, formation of complement-derived membrane attackcomplexes, inflammation, or inflammatory disease. In an in vitro assay,the term “inhibition” may refer to the measurable reduction of somebiological or chemical event, but the person of ordinary skill in theart will appreciate that the measurable reduction need not be total tobe “inhibitory.”

The term “PIC1” refers to a peptide comprising the polar assortant (PA)sequence of IALILEPICCQERAA (SEQ ID NO: 1), as well as peptidescomprising the same amino acid sequence but with modifications such asPEGylation. The term “PIC1 variant” refers to peptides comprising asequence that is at least 85% identical, or at least 90% identical, orat least 95% identical, or at least 99% identical, but not 100%identical, to the PA sequence of IALILEPICCQERAA (SEQ ID NO: 1). PIC1variants may comprise peptides with at least one of the amino acids ofthe PA sequence deleted. PIC1 variants may comprise peptides with anamino acid inserted into the PA sequence. PIC1 variants may comprisepeptides with at least one of the amino acids of the PA sequencesubstituted with another amino acid, such as alanine, a modified aminoacid or an amino acid derivative, such as sarcosine (Sar).

The term “subject” as used herein means any subject for whom diagnosis,prognosis, or therapy is desired. For example, a subject can be amammal, e.g., a human or non-human primate (such as an ape, monkey,orangutan, or chimpanzee), a dog, cat, guinea pig, rabbit, rat, mouse,horse, cattle, or cow.

The term “therapeutically effective amount” as used herein refers to thetotal amount of each active component that is sufficient to show ameaningful patient benefit. The therapeutically effective amount of thepeptide compound varies depending on several factors, such as thecondition being treated, the severity of the condition, the time ofadministration, the route of administration, the rate of excretion ofthe compound employed, the duration of treatment, the co-therapyinvolved, and the age, gender, weight, and condition of the subject,etc. One of ordinary skill in the art can determine the therapeuticallyeffective amount. Accordingly, one of ordinary skill in the art may needto titer the dosage and modify the route of administration to obtain themaximal therapeutic effect.

As used herein, “treat,” “treating,” or “treatment” refers toadministering a therapy in an amount, manner (e.g., schedule ofadministration), and/or mode (e.g., route of administration), effectiveto improve a disorder (e.g., a disorder described herein) or a symptomthereof, or to prevent or slow the progression of a disorder (e.g., adisorder described herein) or a symptom thereof. This can be evidencedby, e.g., an improvement in a parameter associated with a disorder or asymptom thereof, e.g., to a statistically significant degree or to adegree detectable to one skilled in the art. An effective amount,manner, or mode can vary depending on the subject and may be tailored tothe subject. By preventing or slowing progression of a disorder or asymptom thereof, a treatment can prevent or slow deterioration resultingfrom a disorder or a symptom thereof in an affected or diagnosedsubject.

In one aspect is provided a method of inhibiting inflammation in asubject comprising administering a therapeutically effective amount ofPIC1, or a PIC1 variant, to the subject. In another aspect is provided amethod of treating an inflammatory disorder in a subject comprisingadministering a therapeutically effective amount of PIC1, or a PIC1variant, to the subject.

Examples of PIC1 and PIC1 variants include, but are not limited to, thepeptides listed in Table 1.

TABLE 1 Peptide name Peptide sequence PA IALILEPICCQERAA (SEQ ID NO: 1)PA-I1Sar (Sar)ALILEPICCQERAA (SEQ ID NO: 2) PA-A2Sar I(Sar)LILEPICCQERAA(SEQ ID NO: 3) PA-L3Sar IA(Sar)ILEPICCQERAA (SEQ ID NO: 4) PA-I4SarIAL(Sar)LEPICCQERAA (SEQ ID NO: 5) PA-L5Sar IALI(Sar)EPICCQERAA(SEQ ID NO: 6) PA-E6Sar IALIL(Sar)PICCQERAA (SEQ ID NO: 7) PA-P7SarIALILE(Sar)ICCQERAA (SEQ ID NO: 8) PA-I8Sar IALILEP(Sar)CCQERAA(SEQ ID NO: 9) PA-C9Sar IALILEPI(Sar)CQERAA (SEQ ID NO: 10) PA-C10SarIALILEPIC(Sar)QERAA (SEQ ID NO: 11) PA-Q11Sar ALILEPICC(Sar)ERAA(SEQ ID NO: 12) PA-E12Sar IALILEPICCQ(Sar)RAA (SEQ ID NO: 13) PA-R13SarIALILEPICCQE(Sar)AA (SEQ ID NO: 14) PA-A14Sar IALILEPICCQER(Sar)A(SEQ ID NO: 15) PA-A15Sar IALILEPICCQERA(Sar) (SEQ ID NO: 16)dPEG24-PA-dPEG24 dPEG24-IALILEPICCQERAA-dPEG24 (SEQ ID NO: 17) dPEG24-PAdPEG24-IALILEPICCQERAA (SEQ ID NO: 18) PA-dPEG24 IALILEPICCQERAA-dPEG24(SEQ ID NO: 19) PA-dPEG20 IALILEPICCQERAA-dPEG20 (SEQ ID NO: 20)PA-dPEG16 IALILEPICCQERAA-dPEG16 (SEQ ID NO: 21) PA-dPEG12IALILEPICCQERAA-dPEG12 (SEQ ID NO: 22) PA-dPEG08 IALILEPICCQERAA-dPEG08(SEQ ID NO: 23) PA-dPEG06 IALILEPICCQERAA-dPEG06 (SEQ ID NO: 24)PA-dPEG04 IALILEPICCQERAA-dPEG04 (SEQ ID NO: 25) PA-dPEG03IALILEPICCQERAA-dPEG03 (SEQ ID NO: 26) PA-dPEG02 IALILEPICCQERAA-dPEG02(SEQ ID NO: 27) PA-C9SarC10A IALILEPI(Sar)AQERAA (SEQ ID NO: 28)PA-C9SarD10 IALILEPI(Sar)QERAA (SEQ ID NO: 29) PA-P7SarC9SarIALILE(Sar)I(Sar)CQERAA (SEQ ID NO: 30) PA-E6Sar-dPEG24IALIL(Sar)PICCQERAA-dPEG24 (SEQ ID NO: 31) PA-Q11Sar-dPEG24IALILEPICC(Sar)ERAA-dPEG24 (SEQ ID NO: 32) PA-R13Sar-dPEG24IALILEPICCQE(Sar)AA-dPEG24 (SEQ ID NO: 33) PA-A14Sar-dPEG24IALILEPICCQER(Sar)A-dPEG24 (SEQ ID NO: 34) E6SarP7SarIALIL(Sar)(Sar)ICCQERAA (SEQ ID NO: 35) E6SarC9SarIALIL(Sar)PI(Sar)CQERAA (SEQ ID NO: 36) Q11SarP7SarIALILE(Sar)ICC(Sar)ERAA (SEQ ID NO: 37) Q11SarC9SarIALILEPI(Sar)C(Sar)ERAA (SEQ ID NO: 38) R13SarP7SarIALILE(Sar)ICCQE(Sar)AA (SEQ ID NO: 39) R13SarC9SarIALILEPI(Sar)CQE(Sar)AA (SEQ ID NO: 40) A14SarP7SarIALILE(Sar)ICCQER(Sar)A (SEQ ID NO: 41) A14SarC9SarIALILEPI(Sar)CQER(Sar)A (SEQ ID NO: 42) E6AE12A-dPEG24IALILAPICCQARAA-dPEG24 (SEQ ID NO: 43) E6AE12AC9Sar IALILAPI(Sar)CQARAA(SEQ ID NO: 44) E6AE12AP7Sar IALILA(Sar)ICCQARAA (SEQ ID NO: 45)

In some embodiments, PIC1 comprises one or more PEG moieties. The PEGmoieties may be attached to the N-terminus, the C-terminus, or both theN-terminus and C-terminus by PEGylation. In one or more embodiments, 24PEG moieties are attached to the N-terminus. In one or more embodiments,24 PEG moieties are attached to the C-terminus. In one or moreembodiments, 24 PEG moieties are attached to the N-terminus and to theC-terminus. In one or more embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 PEG moietiesare attached to the N-terminus. In one or more embodiments, 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,or 24 PEG moieties are attached to the C-terminus. In one or moreembodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, or 24 PEG moieties are attached to both theN-terminus and the C-terminus.

The PIC1 peptide may be a synthetic peptide. A synthetic peptide isprepared in vitro. Synthetic peptides can be prepared according tovarious methods known in the art. For example, a synthetic peptide canbe prepared by sequentially coupling individual amino acids to form thepeptide. In some embodiments, the carboxyl group of individual aminoacids is sequentially coupled to the amino terminus of a growing peptidechain. Protecting groups can be used to prevent unwanted side reactionsfrom occurring during the coupling process. Peptide synthesis can occurin liquid phase or in solid phase.

Exemplary PIC1 peptides include, but are not limited to, PA-dPEG24 (apeptide comprising the polar assortant (PA) sequence and 24 PEG moietiesat the C-terminus), PA-dPEG20 (comprising 20 PEG moieties at theC-terminus), PA-dPEG16 (comprising 16 PEG moieties at the C-terminus),PA-dPEG12 (comprising 12 PEG moieties at the C-terminus), PA-dPEG08(comprising 8 PEG moieties at the C-terminus), PA-dPEG06 (comprising 6PEG moieties at the C-terminus), PA-dPEG04 (comprising 4 PEG moieties atthe C-terminus), PA-dPEG03 (comprising 3 PEG moieties at theC-terminus), and PA-dPEG02 (comprising 2 PEG moieties at theC-terminus).

PIC1 peptides can inhibit the classical pathway of complement by bindingand blocking activation of the initiating component of the cascade, C1[19, 20]. PA-dPEG24 is a 15-amino acid PEGylated peptide in the PIC1family. PA-dPEG24 can inhibit immune complex-initiated complementactivation as well as inhibit NET formation. PA-dPEG24 can consistentlyinhibit complement activation by a variety of immune complexes and canalso inhibit NET formation initiated by several stimuli.

In some embodiments, PIC1 is effective to inhibit NETosis in thesubject. In some embodiments, the administered PIC1 inhibits MPOactivity in the subject. In various embodiments, the subject is human.NETosis is a process by which neutrophils undergo cell death byreleasing their chromosomal DNA as neutrophil extracellular traps (NET).A NET is web-shaped and is comprised of chromatin fibrils andantimicrobial molecules. In some embodiments, NETosis is stimulated byat least one of a bacterium, a fungus, a parasite or a virus.

PIC1 inhibits MPO-mediated oxidation, as shown in FIG. 16. PA-dPEG24 caninhibit the peroxidase effect of MPO in clinical CF sputum samples exvivo as well as for purified MPO in vitro. PA-dPEG24 also inhibits theperoxidase activity of other heme-based peroxidases including hemoglobinand myoglobin in vitro. PA-dPEG24 can inhibit NET formation in vitro,such as NET formation stimulated by any of phorbol 12-myristate13-acetate (PMA), purified MPO, and immune complex-activated human sera.

PIC1 peptides can be administered to the subject to modulate immunecomplex activation of the complement system and NET formation in adisease. Exemplary diseases include, but are not limited to, LupusNephritis, Serum Sickness, Delayed Type Hypersensitivity (Type IIIHypersensitivity) Reactions, Infective Endocarditis, Auto-immuneGlomerulonephritis, Cryoglobulinemia, Sjogren's syndrome, Small VesselVasculitis, ANCA-associated Vasculitis, Scleroderma and otherinflammatory or autoimmune vasculitis diseases includingglomerulonephropathies, acute respiratory distress syndrome, acute lunginjury, transfusion related acute lung injury, cystic fibrosis, chronicobstructive pulmonary disease (COPD), rheumatoid arthritis,atherosclerosis, Alzheimer's disease, psoriasis, Type 1 DiabetesMellitus, Type 2 Diabetes Mellitus, antiphospholipid antibody syndrome,gout, Crohn's disease, ulcerative colitis, rhabodmyolysis,Obesity/Metabolic Syndrome, Wegener's granulomatosis (WG), thrombosis,systemic inflammatory response syndrome (SIRS), sepsis, retinopathy ofprematurity (ROP), pre-eclampsia, periodontitis, neonatal chronic lungdisease (CLD), necrotizing enterocolitis (NEC), influenza-inducedpneumonitis, inflammatory lung disease (ILD), inflammatory bowel disease(IBD), inflammation in cancer, or bronchopulmonary dysplasia (BPD).

In certain embodiments, PIC1 (e.g., PA-dPEG24) substantially inhibitsNET formation and/or NETosis. In other embodiments, PIC1 (e.g.,PA-dPEG24) inhibits or substantially inhibits NET-mediated inflammatorytissue damage.

In another aspect is provided a method of treating systemic lupuserythematosus (SLE) in a subject comprising administering atherapeutically effective amount of PIC1 to the subject.

Two major aspects of SLE pathogenesis that may be targetedtherapeutically are immune complex-initiated complement activation andneutrophil extracellular trap (NET) formation by neutrophils. Withoutwishing to be bound by theory, the role of anti-C1q antibodies in theblood of SLE patients is an active area of investigation withconsiderable data accumulating to demonstrate a strong associationbetween the presence of anti-C1q antibodies and Lupus Nephritis [7, 8].Investigators have also shown that anti-C1q antibodies from SLE patientsbound to a surface in an ELISA-type assay can activate the classical andlectin pathways [9]. Thus, anti-C1q antibodies may play a role inpathogenesis and in formation of SLE-like immune complexes.

Immune complexes can activate complement generating effectors ofcomplement activation (e.g., C5a, sublytic concentrations of membraneattack complex, etc.) that interact with and can stimulate humanneutrophils [10-13]. However, articles describing that immune complexescan induce neutrophils to generate NETs have focused on the role of Fcreceptors in this process [14-17]. If the articles show a link betweenimmune complexes and NETs, the contribution of complement activation inthis process remains unclear. Akong-Moore et al. [18] suggested that amajor pathway of NET formation can occur via MPO and its primaryfunction of generating hypochlorous acid from hydrogen peroxide andchloride ion. NET formation may be blocked by utilizing an MPOinhibitor.

In some embodiments, PIC1 is effective to inhibit NETosis in thesubject. In some embodiments, NETosis is stimulated by at least one of abacterium, a fungus, a parasite or a virus. PIC1 can be administered tomodulate immune complex complement activation and NET formation. In oneembodiment, the PIC1 is PA-dPEG24. In one embodiment, PIC1 can be usedto modulate C1-antiC1q immune complexes. In one embodiment, PIC1 can beused to inhibit immune complex activation and NET formation. In certainembodiments, PIC1 can be used to limit the generation ofpro-inflammatory complement effectors. In one embodiment, PIC1 can beused to limit the generation of C5a and sC5b-9. In certain embodiments,PIC1 inhibits NET formation by human neutrophils stimulated by PMA.

In some embodiments, the administered PIC1 inhibits MPO activity in thesubject. PIC1 can be used to inhibit NET formation by human neutrophilsstimulated by MPO. In accordance with certain aspects, PIC1 can be usedto inhibit NET formation by human neutrophils stimulated by immunecomplex activated sera. In accordance with certain aspects, PIC1 isdelivered parenterally. In various embodiments, the subject is human.

In accordance with certain aspects, PIC1 can be used to modulate immunecomplex complement activation and neutrophil formation to treat SLE,Lupus Nephritis, Serum Sickness, Delayed Type Hypersensitivity (Type IIIHypersensitivity) Reactions, Infective Endocarditis, Auto-immuneGlomerulonephritis, Cryoglobulinemia, Sjogren's syndrome, Small VesselVasculitis, ANCA-associated Vasculitis, Scleroderma and otherinflammatory or autoimmune vasculitis diseases, Glomerulonephropathies,Acute Respiratory Distress Syndrome, Acute Lung Injury, TransfusionRelated Acute Lung Injury, Cystic Fibrosis, Chronic ObstructivePulmonary Disease, Rheumatoid Arthritis, Atherosclerosis, Alzheimer'sDisease, Psoriasis, Type 1 Diabetes Mellitus, Type 2 Diabetes Mellitus,Antiphospholipid Antibody Syndrome, Gout, Crohn's Disease, UlcerativeColitis, Rhabodmyolysis, and Obesity/Metabolic Syndrome.

The experiments of Example 1 demonstrate that PA-dPEG24 can inhibitimmune complex-initiated complement activation and the generation ofpro-inflammatory complement effectors. This suggests that complementinhibitory peptides could moderate aspects of pathogenesis in diseaseswhere immune complex activation of the complement system plays a vitalrole, such as SLE, Lupus Nephritis, Serum Sickness, Delayed TypeHypersensitivity (Type III Hypersensitivity) Reactions, InfectiveEndocarditis, Auto-immune Glomerulonephritis, Cryoglobulinemia,Sjogren's syndrome, Small Vessel Vasculitis, ANCA-associated Vasculitis,Scleroderma and other inflammatory or autoimmune vasculitis diseases,Glomerulonephropathies, Acute Respiratory Distress Syndrome, Acute LungInjury, Transfusion Related Acute Lung Injury, Cystic Fibrosis, ChronicObstructive Pulmonary Disease, Rheumatoid Arthritis, Atherosclerosis,Alzheimer's Disease, Psoriasis, Type 1 Diabetes Mellitus, Type 2Diabetes Mellitus, Antiphospholipid Antibody Syndrome, Gout, Crohn'sDisease, Ulcerative Colitis, Rhabodmyolysis, and Obesity/MetabolicSyndrome. Additionally, C1 and anti-C1q antibodies can be utilized in anovel immune complex to model a type of immune complex that could bepredicted to be formed in the plasma of SLE patients with anti-C1qantibodies. PA-dPEG24 also blocked complement activation by C1-antiC1qimmune complexes, consistent with the other immune complex types tested.

Immune complex-initiated complement-activated human sera can initiateNET formation, e.g., as demonstrated in Example 1. The result issurprising and stands in contrast to prior observations that immunecomplexes by themselves can initiate NET formation via Fc receptors[14-17]. Under the experimental conditions of Example 1 the contributionof immune complex-initiated complement activation to NET formation wasmuch greater than that of immune complexes alone. Without wishing to bebound by theory, immune complex-initiated complement activation may bean important mechanism of NET formation in SLE given the presence ofimmune complexes in active SLE disease.

The experiments in Example 1 also show that PA-dPEG24 can inhibit NETformation by human neutrophils initiated by PMA, MPO or immunecomplex-initiated complement-activated human sera. Without wishing to bebound by theory, PA-dPEG24 blocks NET formation by inhibiting theMPO-mediated pathway based at least on an ability of PMA to stimulateNET formation via an MPO-mediated pathway [18] and the results describedherein utilizing purified MPO. The ability of PA-dPEG24 to inhibit NETformation after immune complex activation of human sera has occurredsuggests that NET formation may occur by blocking the MPO-mediatedpathway. PA-dPEG24 peptide can block both classical pathway complementactivation and inhibit NETosis. PA-dPEG24 and other PIC1 proteins couldbe useful in methods of treating SLE by acting upon two currentlyuntargeted aspects of SLE pathogenesis.

In another aspect is provided a method of treating transfusion-relatedacute lung injury (TRALI) in a subject comprising administering atherapeutically effective amount of PIC1 to the subject.

Transfusion-related acute lung injury (TRALI) is a disease ofrespiratory distress initiated by blood transfusion and the leadingcause of transfusion-related death. Described herein is a novel‘two-hit’ rat model of TRALI utilizing mismatched erythrocytes thatcause neutrophil infiltration of lung parenchyma. The two-hit rat modelallows for assessing the role of peptide inhibitor of complement C1(PIC1) in attenuating lung injury in this new TRALI model.

Described herein is a novel model of TRALI, with data showing theability of PIC1 to attenuate TRALI-mediated disease in this model. Anumber of ‘two-hit’ models, utilizing LPS as the ‘first-hit’ and eitheran antibody-dependent (e.g. H2K^(d), HLA, etc.) or anantibody-independent (e.g. aged RBCs, lysoPCs, RBC supernatants, etc.)stimulus as the ‘second hit’ have been published in the literature in avariety of species. The model described herein is unique in that it usesantibody-initiated complement-mediated hemolysis of transfusederythrocytes as the ‘second hit’, in contrast to transfusion of RBCsusing syngeneic erythrocytes. Wistar rats possessing preexistingantibodies to the A antigen of human RBCs can initiate classicalcomplement activation that can lead to a vigorous intravascularhemolysis after transfusion of type A or type AB human erythrocytes.

PIC1 is a multifunctional molecule. In an AIHTR model, PIC1 can inhibithemolysis of transfused mismatched RBCs by suppressing classicalcomplement pathway activation and RBC lysis by the membrane attackcomplex (MAC). PIC1 can act as an antioxidant molecule in vitro toinhibit peroxidase activity of free heme, hemoglobin and myoglobin aswell as the peroxidase activity of MPO.

In some embodiments, the PIC1 is administered before the subject isadministered a blood transfusion, after the subject is administered theblood transfusion, and/or during the blood transfusion.

In some embodiments, PIC1 is effective to inhibit NETosis in thesubject. In various embodiments, the subject is human. In someembodiments, NETosis is stimulated by at least one of a bacterium, afungus, a parasite or a virus.

In some embodiments, the administered PIC1 inhibits MPO activity in thesubject. Furthermore, PIC1 can inhibit NETosis mediated by MPO. Withoutwishing to be bound by theory, PIC1 may reduce the massive infiltrationof neutrophils into the lung tissue and may attenuate TRALI byinhibiting complement activation and preventing the generation of theanaphalotoxins C3a and C5a as well as hemoglobinemia throughMAC-mediated hemolysis (FIG. 15). PIC1 can attenuateperoxidase-generated ROS activity from free hemoglobin and MPO as wellas NETosis in vitro.

Currently there are no pharmacological interventions to treat TRALI withcurrent standard of care consisting of mechanical ventilation andhemodynamic support. The animal model described herein may model atrauma-like clinical scenario in which the patient requires massivetransfusion of packed RBCs that leads to TRALI. The ability of PIC1 toblock TRALI-like pathogenesis from LPS infusion followed by mismatchedRBCs by inhibiting classical pathway complement activation, MPO-mediatedROS formation as well as NETosis suggests that PIC1 may have potentialas a pharmacological agent to mitigate multiple aspects of TRALIpathogenesis.

Intranasal administration of DNase reduced symptoms of TRALI in animalmodels, suggesting that inhibition of NET formation could be a usefultherapeutic strategy. A major pathway of NET formation can be mediatedby neutrophil-generated MPO through its primary role in producinghypochlorous acid from chloride ion and hydrogen peroxide as well asMPO-derived reactive oxygen species. Phorbol 12-mystate 13-acetate(PMA)-stimulated NET formation can be inhibited by the MPO inhibitorABAH in vitro revealing another potential mechanism by which to inhibitNET formation.

In a rat model of acute intravascular hemolytic transfusion reaction(AIHTR) utilizing transfusion of mismatched erythrocytes, the ratspecies has preexisting antibodies to the A antigen of humanerythrocytes, resulting in a robust AITHR after transfusion of humanerythrocytes from a type A or type AB donor. This AIHTR model may causeantibody-initiated classical complement pathway activation producingacute kidney injury and a highly inflammatory systemic response. Thepathogenic aspects of antibody-initiated complement activation andmobilization of neutrophils suggests that this model can adapted toyield a TRALI phenotype, if the inflammatory response were directedtowards the lungs.

Antibody-mediated activation of the complement system is directed by theclassical complement pathway in which the initiating complex, C1, isbound by IgM or multiple IgG triggering activation and downstreameffector functions (i.e., C3a, C5a and membrane attack complexformation). PIC1 peptide inhibitors of the classical complement pathwaycan bind C1q, the recognition molecule of the C1 complex, to preventantibody-mediated activation. The PIC1 derivative PA-dPEG24(IALILEPICCQERAA-dPEG24 (SEQ ID NO: 19)) has been demonstrated toinhibit classical pathway activation both in vitro and in vivo whenadministered intravascularly into rats where it can achieve >90%systemic inhibition of complement activation by 30 seconds. While PIC1derivatives have been previously shown to inhibit classical complementpathway-mediated ABO-mismatched hemolysis in vitro, PIC1 can inhibitantibody-initiated complement-mediated hemolysis in a rat model of AIHTRwhen administered either immediately before or after transfusion of themismatched erythrocytes. Additionally, PIC1 has also been shown toinhibit the peroxidase activity of MPO and NETosis in vitro. A unique,mismatched erythrocyte-based, ‘two-hit’ TRALI model has been developedand demonstrated that prophylactic administration of PIC1 mitigatesacute lung injury and other hallmarks of this respiratory syndrome.

EXAMPLES

The present invention is also described and demonstrated by way of thefollowing examples. However, the use of this and other examples anywherein the specification is illustrative only and in no way limits the scopeand meaning of the invention or of any exemplified term. Likewise, theinvention is not limited to any particular preferred embodimentsdescribed here. Indeed, many modifications and variations of theinvention may be apparent to those skilled in the art upon reading thisspecification, and such variations can be made without departing fromthe invention in spirit or in scope. The invention is therefore to belimited only by the terms of the appended claims along with the fullscope of equivalents to which those claims are entitled.

Example 1

This example describes testing of Peptide Inhibitor of Complement C1(PIC1) in in vitro assays of immune complex-mediated complementactivation in human sera and assays for NET formation by humanneutrophils.

Blood from healthy donors was obtained. PA-dPEG24(IALILEPICCQERAA-dPEG24 (SEQ ID NO: 19)) was manufactured by PolyPeptideGroup (San Diego, Calif.) to ≥95% purity verified by HPLC and massspectrometry analysis. Lyophilized PA-dPEG24 was solubilized in normalsaline with 0.01 M Na₂HPO₄ buffer to 37.5 mM. Purified MPO was purchasedfrom Lee Biosolutions (Maryland Heights, Mo.). Intravenous ImmuneGlobulin was purchased from Baxter Healthcare Corporation (WestlakeVillage, Calif.), Ovalbumin from Sigma Aldrich (St Louis, Mo.), and theAnti-ovalbumin antibody from Abcam (Cambridge, Mass.). Goat anti-C1q andhuman C1 were purchased from Complement Technology (Tyler Tex.). PMA(Phorbol 12-myristate 13-acetate) and hydrogen peroxide were purchasedfrom Fisher Scientific (Hampton, N.H.).

The complement permissive GVBS⁺⁺ buffer was veronal-buffered saline with0.1% gelatin, 0.15 M CaCl₂, and 1 mM MgCl₂ [29]. The complementinhibitory buffer GVBS⁻⁻ was a veronal-buffered saline with 0.1% gelatinand 10 mM EDTA. Pooled normal human serum (NHS) was prepared aspreviously described [29].

Immune complex activation of NHS was performed as follows. NHS wasstimulated with three different types of immune complexes (IC) to inducecomplement activation. Heat-aggregated IgG was generated by incubatingintravenous immune globulin at 50 mg/ml at 63° C. for 30 min [26].Ovalbumin-antiovalbumin immune complexes were made by incubating 0.01 mlanti-ovalbumin antibody with an equal volume of ovalbumin, at 0.25mg/nil, at 37° C. for 30 minutes and then storing at 4° C. overnight. C1immune complexes were formed by incubating 0.02 ml anti-C1q goat serawith 5 μl of C1, at 200 μg/ml, at 30° C. for 30 minutes and then placingin an ice water bath. For C5a and C5b-9 assays, activation of NHS wasperformed by pre-incubating 5% NHS with titrating concentrations ofPA-dPEG24 in 0.3 ml of GVBS' buffer for 30 minutes at room temperature.Then 2 ml of either heat-aggregated IVIg, or ovalbumin IC, or 5 μI ofC1-antiC1q IC was added to the mix for 30 min. at 37° C. This reactionwas stopped with the addition of an equal volume of GVBS⁻⁻. For iC3bdetection, 1% NHS was used and the rest of the protocol remained thesame.

ELISA was performed as follows. Samples were assayed using C5a, iC3b,and SC5b-9 ELISAs. A C5a ELISA kit (R&D Systems) was used per themanufacturer's instructions. ELISAs for iC3b and SC5b-9 were performedas previously described [30]. In iC3b ELISA, a goat anti-human C3antibody (Complement Technology, Tyler Tex.) was used in for capture, amouse anti-human iC3b antibody (Quidel, San Diego Calif.) for probing,and a goat anti-mouse HRP antibody for detection. In SC5b-9 ELISA, arabbit anti-human SC5b-9 antibody (Complement Technology) was used forcapture, a mouse anti-human SC5b-9 monoclonal antibody (Quidel) forprobing, and a chicken anti-mouse HRP antibody for detection.Colorimetric detection was performed with TMB, stopped with H₂SO₄ andread on a BioTek Synergy HT plate reader at 450 nm.

Neutrophils from the blood of healthy volunteers were purified fromheparinized blood by Hypaque-Ficoll step gradient centrifugation,dextran sedimentation, and hypotonic lysis, as previously described[31].

A Neutrophil Extracellular Trap assay was performed in a microtiterplate as follows. The formation of NETs was induced by incubating2.0×10⁵ neutrophils in a 96 well tissue culture plates with RPMI mediaalone, or adding 0.05% of H₂O₂, or 12 nM PMA, or 8 μg/ml MPO, orPA-dPEG24 at various concentrations. For immune complex sera induced NETformation, activated sera was made by adding 5 μl ofovalbumin-anti-ovalbumin immune complex to 5% NHS in 0.3 ml of GVBS′.This combination was allowed to incubate for 30 minutes at 37° C., andthen 0.05 ml was added to the neutrophils in RPMI. Cells were thenincubated for 1.5 hours at 37° C. in 5% CO₂ incubator.

NET formation was quantitatively assayed as follows. Free DNA wasmeasured by PicoGreen in the supernatant recovered from the NETmicrotiter plate well assay [18]. Five hundred units of monococcalnuclease (Fisher) were added to each well to allow for digestion ofreleased extracellular DNA for 10 minutes in 37° C. incubator. Thepreparation was then aliquoted into an adjacent well and mixed 1:1 withprepared PICO green reagent (Fisher). The fluorescence was thenquantified on a BioTek microplate reader at Excitation 485 nm/Emission528 nm.

NET formation was assayed by fluorescence microscopy. Purified humanneutrophils were assayed on a glass slide as follows. Cells werecombined with RPMI media and the indicated stimuli as mentioned above ina tube and then aliquoted onto a glass slide circled with a hydrophobicslide marker. The slides were incubated for 1.5 hours at 37° C. in a 5%CO₂ incubator at 37 degrees for 1.5 hours. Slides were fixed overnightwith 4% paraformaldehyde at 4° C.

For all staining, the following conditions were used. Slides were washedin PBS and incubated in blocking solution (2% normal goat serum+2%bovine serum albumin in PBS) for 1 hour at room temperature. Then theslides were incubated with primary antibody at 1:300 in 2% BSA in PBSfor 1 hour at room temperature. Slides were washed in PBS 3 times andincubated in fluorescent-labeled secondary antibody at 1:1000 or DAPI(Southern Biotech) at 0.25 pg/ml final in 2% BSA in PBS for 1 hour atroom temperature. Slides were then washed 3 times in PBS and wereimaged. Cells were visualized using a DP70 Digital Camera (OlympusCenter, Valley Forge, Pa.), mounted on a BX50, Olympus microscope.Staining antibody pairs used were rabbit anti-MPO (Thermo Scientific)and rabbit anti-Histone H3 (Abcam) with the secondary goat anti-rabbitAlexa Fluor 488 (Novus Biologicals). Also, mouse anti-elastase(Invitrogen) was used with the secondary goat anti-mouse Alexa Fluor 568(Novus Biologicals).

Statistical analysis was performed as follows. Quantitative data wereanalyzed determining means, standard error (SEM), and Student's t-test[32] using Excel (Microsoft, Redmond, Wash.).

PA-dPEG24 was shown to inhibit immune complex-initiated complementactivation. To evaluate the ability of PA-dPEG24 to inhibit immunecomplex-initiated complement activation, the archetypal immune complexstimulant of heat-aggregated IgG [25, 26] was utilized in pooled normalhuman serum (NHS). Three important effectors resulting from complementactivation were assayed: the major pro inflammatory anaphylatoxin, C5a,a cleavage product of C3 activation, iC3b, and the membrane attackcomplex, C5b-9. The data is shown in FIGS. 1A-1C. FIG. 1A shows thatPA-dPEG24 inhibits C5a generation in normal human serum (NHS) stimulatedwith heat-aggregated IgG (Agg-IgG) immune complexes. Five independentexperiments were performed, with the SEM shown. FIG. 1B shows PA-dPEG24inhibition of iC3b generation in normal human serum (NHS) stimulatedwith heat-aggregated IgG (Agg-IgG) immune complexes. Four independentexperiments were performed, with the SEM shown. FIG. 1C shows PA-dPEG24inhibition of SC5b-9 generation in normal human serum (NHS) stimulatedwith heat-aggregated IgG (Agg-IgG) immune complexes. Six independentexperiments were performed, with the SEM shown.

For each assay, PA-dPEG24 dose-dependently inhibited elaboration of theeffector after stimulation with heat-aggregated IgG compared with noinhibitor. Statistically significant inhibition was achieved in eachassay at ≥0.5 mM PA-dPEG24 (P<0.05). For C5a, 1 mM PA-dPEG24 lead to a61% reduction (P=0.002) compared with heat-aggregated IgG with noinhibitor. These results suggest that PA-dPEG24 can inhibit immunecomplex-initiated complement activation in human sera.

To provide confirmation of the results with heat-aggregated IgG, theantigen-antibody immune complex most often utilized in animal models ofcomplement activation, ovalbumin and antiovalbumin [27, 28], was thentested. The ovalbumin-antiovalbumin immune complexes were used tostimulate complement activation in NHS and the same three effectors,C5a, iC3b and C5b-9, were measured. PA-dPEG24 dose dependently inhibitedgeneration of each complement effector with statistically significantinhibition achieved at ≥0.25 mM PA-dPEG24 (P<0.03) compared withovalbumin-antiovalbumin alone. The data is shown in FIGS. 2A-2C. FIG. 2Ashows PA-dPEG24 inhibition of C5a generation in normal human serum (NHS)stimulated with ovalbumin-antiovalbumin immune complexes. Fourindependent experiments were performed, with the SEM shown. FIG. 2Bshows PA-dPEG24 inhibition of iC3b generation in normal human serum(NHS) stimulated with ovalbumin-antiovalbumin immune complexes. Fourindependent experiments were performed, with the SEM shown. FIG. 2Cshows PA-dPEG24 inhibition of SC5b-9 generation in normal human serum(NHS) stimulated with ovalbumin-antiovalbumin immune complexes. At 2 mMPA-dPEG24 the measured SC5b-9 was at the lower limit of detection. Threeindependent experiments were performed, with the SEM shown. Theseresults provide additional confidence that PA-dPEG24 can inhibit immunecomplex-initiated complement activation in human sera.

Due to the importance of anti-C1q antibodies in a subset of patientswith SLE, immune complexes with human C1 and anti-C1q antibodies (goat)were generated. These immune complexes activated NHS leading to robustgeneration of C5a, iC3b and SC5b-9. The data is shown in FIGS. 3A-3C.

FIG. 3A shows PA-dPEG24 inhibition of C5a generation in normal humanserum (NHS) stimulated with C1-antiC1q immune complexes. Fourindependent experiments were performed, with the SEM shown. FIG. 3Bshows PA-dPEG24 inhibition of iC3b generation in normal human serum(NHS) stimulated with C1-antiC1q immune complexes. Four independentexperiments were performed, with the SEM shown. FIG. 3C shows PA-dPEG24inhibition of SC5b-9 generation in normal human serum (NHS) stimulatedwith C1-antiC1q immune complexes. Four independent experiments wereperformed, with the SEM shown.

PA-dPEG24 dose-dependently inhibited C1-antiC1q generation of C5a in NHSat each concentration (P≤0.03). C1-antiC1q generation of iC3b wasinhibited by 2 mM PA-dPEG24 (P<0.02) to a level similar to NHS baseline.PA-dPEG24 dose-dependently inhibited C1-antiC1q generation of SC5b-9 forconcentrations ≥0.13 mM (P<0.03). These results show that PA-dPEG24 caninhibit C1-antiC1q immune complex-initiated complement activation inhuman sera.

PA-dPEG24 was shown to inhibit PMA-initiated NET formation by humanneutrophils. To evaluate whether PA-dPEG24 can inhibit neutrophilextracellular trap (NET), purified human neutrophils and the commonlyutilized stimulus phorbol 12-mystate 13-acetate (PMA) were used in amanner similar to methods described by Akong-Moore et al [18].Extracellular DNA and myeloperoxidase (two major components of NETs)were visualized with DNA and anti-MPO antibody, respectively.

Human neutrophils stimulated with PMA and hydrogen peroxide generatedmany NETs visualized by fluorescence microscopy of extracellular DNA andextracellular MPO. FIG. 4 shows PA-dPEG24 inhibition of PMA-initiatedNET formation with human neutrophils (PMN) assayed by fluorescencemicroscopy and PicoGreen quantitation of free DNA. The first row showsunstimulated neutrophils, the second row shows neutrophils stimulatedwith PMA and hydrogen peroxide (H₂O₂) and third row shows neutrophilsstimulated with PMA+11202 in the presence of 5 mM PA-dPEG24 (PIC1). Thefirst column are slides probed with DAPI to visualize DNA and the secondcolumn are slides probed with anti-MPO antibody. The graph showsPA-dPEG24 (5 mM) inhibition of NET generation by human neutrophilsstimulated with PMA+11202 assayed by PicoGreen. Three independentexperiments were performed, with the SEM shown.

In the presence of 5 mM PA-dPEG24, PMA and hydrogen peroxide did notgenerate NETs that could be identified by fluorescence microscopy. NETformation was then quantified by measuring free DNA in a PicoGreen-basedassay from supernatants of human neutrophils stimulated in microtiterplate wells. PA-dPEG24 (5 mM) was able to inhibit free DNA elaborationby 2.6-fold (P=0.01) in the presence of PMA and hydrogen peroxidecompared with no inhibitor (FIG. 4). This reduction for PA-dPEG24 was toa level similar to baseline without PMA.

Fluorescence microscopy was also performed utilizing the sameexperimental conditions, but instead additional NET constituentsextracellular neutrophil elastase and histone H3 were probed. FIG. 5shows PA-dPEG24 inhibition of PMA-initiated NET formation with humanneutrophils (PMN) assayed by fluorescence microscopy for DNA (DAPI),neutrophil elastase (αNE), and histone H3 (αhistone). The first rowshows unstimulated neutrophils, the second row shows neutrophilsstimulated with PMA and hydrogen peroxide (H₂O₂) and third row showsneutrophils stimulated with PMA+H₂O₂ in the presence of 5 mM PA-dPEG24(PIC1). The first column are slides probed with DAPI to visualize DNA,the second column are slides probed with anti-neutrophil elastaseantibody and the third row is probed with anti-histone H3 antibody.Representative images are shown.

The above show that stimulation with PMA and hydrogen peroxide resultedin copious NET formation, which was inhibited in the presence ofPA-dPEG24 (5 mM). These results suggest that PA-dPEG24 can inhibitPMA-stimulated NET formation by human neutrophils.

PA-dPEG24 was shown to inhibit MPO-initiated NET formation by humanneutrophils. Akong-Moore et al [18] suggest that MPO is a criticalmediator in PMA-stimulated NET formation, however this was never testedusing purified MPO. The above experiments with purified humanneutrophils were repeated but with purified MPO substituted for PMA asthe stimulus for NET formation. Neutrophil stimulation with purified MPOand hydrogen peroxide caused extensive NET formation visualized by DAPIstaining and anti-MPO staining. FIG. 6 shows PA-dPEG24 inhibition ofMPO-initiated NET formation with human neutrophils (PMN) assayed byfluorescence microscopy and PicoGreen quantitation of free DNA. Thefirst row shows unstimulated neutrophils. The second row showsneutrophils stimulated with MPO and hydrogen peroxide (H₂O₂). The thirdrow shows neutrophils stimulated with MPO+H₂O₂ in the presence ofPA-dPEG24 (PIC1) and the fourth row shows neutrophils incubated withPA-dPEG24 (PIC1) only. The first column shows slides probed with DAPI tovisualize DNA. The second column shows slides probed with anti-MPOantibody. The graph shows PA-dPEG24 inhibition of NET generation byhuman neutrophils stimulated with MPO+hydrogen peroxide assayed byPicoGreen. Three independent experiments were performed, with the SEMshown.

NET formation in presence of MPO and hydrogen peroxide was blocked withPA-dPEG24 (5 mM). Neutrophils incubated with PA-dPEG24 alone appearednormal by fluorescence microscopy. When NET formation was quantified byPicoGreen measurement, 1.1 mM of PA-dPEG24 lead to a 30% (P=0.02)reduction in free DNA and 4.5 mM of PA-dPEG24 resulted in a 3.7-fold(P=0.001) reduction in free DNA compared with stimulation with MPO, butno inhibitor (FIG. 6). In the presence of MPO plus 4.5 mM PA-dPEG24,measured free DNA was not statistically different from unstimulatedneutrophils. These results suggest that PA-dPEG24 inhibits NET formationvia the MPO-mediated pathway.

PA-dPEG24 inhibits immune complex-initiated NET formation by humanneutrophils. A potential relationship between immune complex-initiatedcomplement-activated human sera and NET formation by human neutrophilswas evaluated. Complement in normal human sera was activated withovalbumin-antiovalbumin immune complexes, as was performed in theexperiments of FIG. 2. The immune complex-initiated complement-activatedhuman sera was then incubated with purified human neutrophils resultingin NET formation quantified by free DNA measurement with PicoGreen. Therelative contribution of immune complexes was initially evaluated bythemselves compared with immune complex-initiated complement-activatedhuman sera for generation of NETs. The presence of immune complexes bythemselves did not significantly (P=0.39) increase NET formationcompared with neutrophils alone (FIG. 7A). However, immune complexactivation of complement in sera increased NET formation >20-fold(P=0.009) compared with immune complexes alone after subtracting thebackground. These results demonstrate for the first time that immunecomplex-initiated complement-activated sera is a strong stimulus for NETformation.

Hydrogen peroxide was tested to determine whether it further enhancedNET formation by immune complex-activated sera, with hydrogen peroxideapproximately doubling the signal (FIG. 7B). Testing of PA-dPEG24inhibition of NETosis was performed with PA-dPEG24 added aftercomplement activation of the sera by immune complexes had already beenallowed to occur. Therefore, any effect of PA-dPEG24 on NETosis happeneddownstream of complement activation. In the presence of immunecomplex-activated sera and hydrogen peroxide, 2.2 mM PA-dPEG24 decreasedfree DNA by 23% (P=0.037) and 4.5 mM PA-dPEG24 decreased free DNA by3-fold (P<0.001) compared with no inhibitor (FIG. 7B). These conditionswere also visualized by fluorescence microscopy (FIG. 7C). In thepresence of PA-dPEG24 (5 mM) and immune complex-initiatedcomplement-activated sera no NETs were identified by fluorescencemicroscopy.

These findings suggest that immune complex activated human sera canstimulate human neutrophils to form NETs and that this can be inhibitedwith PA-dPEG24. Inhibition of NETosis by PA-dPEG24 was a surprisingfinding given that the inhibition of NET formation occurred aftercomplement activation had occurred in the serum and thus the initiatingstimulus was unaffected. These results suggest the novel idea thatimmune complex-initiated complement-activated sera may cause NETosis viaan MPO-mediated pathway, which was blocked with PA-dPEG24. Takentogether, these experiments consistently show that PA-dPEG24 can inhibitNET formation by human neutrophils initiated by a variety of stimulants.

In conclusion, the lead PIC1 derivative, PA-dPEG24(IALILEPICCQERAA-dPEG24 (SEQ ID NO: 19)), was able to dose-dependentlyinhibit complement activation initiated by multiple types of immunecomplexes, including C1-antiC1q immune complexes, limiting thegeneration of pro-inflammatory complement effectors including C5a andmembrane attack complex (sC5b-9). PA-dPEG24 was also able todose-dependently inhibit NET formation by human neutrophils stimulatedby phorbol 12-mystate 13-acetate (PMA), myeloperoxidase (MPO) or immunecomplex activated human sera. These results suggest that PA-dPEG24inhibition of NETs occurs by blocking the MPO pathway of NET formation.Together these results demonstrate that PA-dPEG24 can inhibit immunecomplex activation of the complement system and NET formation,suggesting that PIC1 peptides could be used as a therapeutic approach tomodulate these two critical aspects of SLE pathogenesis that are notaddressed by current pharmacological treatments.

Example 2

In this Example, Wistar rats were primed with lipopolysaccharidefollowed by 30% transfusion of mismatched erythrocytes, against whichthe rats have preexisting antibodies. Sham and vehicle animals were usedas controls with a subgroup of animals receiving PIC1 two minutes beforetransfusion. At 4 hours, blood was isolated for complete blood count.Isolated lung tissue was stained with hematoxylin and eosin andmyeloperoxidase (MPO) activity in lung tissue was quantified in afunctional assay. Free DNA in plasma was detected by PicoGreen staining.

This novel ‘two-hit’ model utilizing erythrocyte transfusion yielded arobust TRALI phenotype. Compared to vehicle controls, lungs of PIC1treated animals showed reduced lung damage, neutrophil invasion and MPOactivity in the lung tissue. Additionally, rats receiving PIC1demonstrated a reduction of free DNA in the blood suggestive ofattenuated neutrophil extracellular trap formation previously associatedwith TRALI. The results shown below demonstrate that PIC1 attenuatesacute lung injury in a novel animal model of TRALI.

Adolescent male Wistar rats (200-250 g) were purchased from Hilltop LabAnimals with indwelling jugular catheters and used under EasternVirginia Medical School (EVMS) IACUC (Institutional Animal Care and UseCommittee) approved protocols.

Healthy human volunteer donated AB blood used to generate purified humanred blood cells (RBCs) was obtained after written informed consent underan EVMS approved Institutional Review Board protocol (EVMS IRB #02-06-EX0216). Human RBCs acquired the morning of the animal experiments wereprocessed as described previously. Human blood was purified on aHistopaque gradient by centrifugation. The RBCs were then separated fromwhite blood cells and platelets and resuspended in saline. Rats (200 g)have a nominal circulating blood volume of 14 mL with a nominal 40%hematocrit. For transfusion, approximately 2 mL of human RBC at 80%hematocrit was transfused, which results in approximately 30%transfusion to the rats.

Plasma generated from the above experiments was analyzed for freehemoglobin using spectrophotometry, as described previously. Donorerythrocytes were hemolyzed with water to generate a standard curve fromwhich the amount of hemolyzed erythrocytes in each sample was calculatedwith respect to the free hemoglobin measurements.

Flow cytometry was performed using a FACSCalibur flow cytometer (BectonDickinson, Franklin Lakes, N.J., USA) with DXP 8 Color 488/637/407upgrade (Cytek Development, Freemont, Calif., USA). The data wasacquired using Cytek FlowJo CE version 7.5.110.6. Approximately 1×10⁵events, selected for erythrocytes, per sample were gathered for singlelabeled flow, respectively. Data was analyzed using FlowJo X version10.0.7r2 (FlowJo LLC).

For single labeled flow, the cells collected after separating the plasmawere washed, diluted and stained with FITC-conjugated anti-human CD235a(glycophorin A, eBioscience) at 1:200 in GVBS (veronal-buffered saline(VBS) with 0.1% gelatin, 0.01 mol/L EDTA (ethylenediaminetetraaceticacid)) for 20 min while shaking at room temperature to minimizeagglutination. An antibody control consisted of mouse IgG2b Iso-controlFITC at 1:200 (eBioscience).

Lung tissue stained with H&E was analyzed by a clinician blinded to theexperimental groups (vehicle only and PIC1-treated animals). Neutrophilinfiltration and cell wall thickening were scored on a scale of 0-4:0=normal lungs, 1=minor lung involvement, 2=moderate lung involvement,3=serious lung involvement, 4=severe lung involvement.

A myeloperoxidase (MPO) activity assay was conducted as follows. Frozenlung tissue was diced and homogenized on ice in 50 mM potassiumphosphate, and centrifuged at 10,000 RPM at 4° C. for 15 minutes afterwhich the supernatant was discarded. To solubilize MPO from theneutrophils in lung tissue, the pellet was resuspended in 500 ml 50 mMhexadecyltrimethylammonium bromide (HTAB), homogenized by sonication andsnap frozen in liquid nitrogen. This process was repeated twice andsamples were then centrifuged at 10,000 RPM at 4° C. for 10 minutes andsupernatant collected.

Peroxidase activity in these samples was measured using10-actyl-3,7-dihydroxyphenoxazine (ADHP, Amplex Red) following MPOantibody capture from samples as previously described. Fluorescence wasread from 0 to 600 seconds in twenty-five second intervals whichprovided twenty-five data points per well at an excitation wavelength of535 nm and emission wavelength of 590 nm using a BioTek microplatereader. Purified MPO was used as a positive control and phosphatebuffered saline (PBS) as a negative control. All activity assays wereperformed in triplicate.

Free DNA was measured by PicoGreen in rat plasma as previouslydescribed. Briefly, plasma samples were diluted 1:10 in 10 mM Tris-HCl,1 mM EDTA, pH 8.0 (TE) buffer and 50 uL of each sample was added to thewells along with 50 uL of a 1:200 dilution of PicoGreen (LifeTechnologies) and incubated at room temperature for 10 minutes,protected from light. A DNA standard curve was prepared in TE Buffer.The fluorescence was then read at an excitation wavelength of 485 nm andan emission wavelength of 520 nm using a BioTek microplate reader. Allfree DNA measurements were done in triplicate.

Means and standard errors were calculated from independent experimentsand statistical comparisons were made using Student t test (MicrosoftExcel XP).

In a rat model of acute intravascular hemolytic transfusion reaction(AIHTR), transfusion of 15% mismatched RBCs resulted in intravascularhemolysis and acute kidney damage. In this model, naturally circulatinganti-A antibodies in Wistar rats initiate classical complementactivation and hemolysis of the transfused erythrocytes. To ascertain ifthe AIHTR model could be adapted to mimic a TRALI phenotype, ascendingdoses of mismatched RBC transfusions (15, 30 or 45%) were initiallytested and it was determined that a 30% transfusion produces nearmaximal amounts of complement-mediated hemolysis (FIG. 8A). Saturationat 45% transfusion suggests that the amount of antibody binding theerythrocytes with sufficient clustering to initiation complementactivation has likely been exceeded. The 30% transfusion produced anintermediate phenotype of RBC survival compared to the 15 and 45%transfusions as assessed by flow cytometry (FIG. 8B). At 4 hours posttransfusion, no histological lung damage occurred, even at 45%transfusion (FIGS. 9A-9C).

A significant increase in white blood cells (WBC) (P=1.08×10⁻⁶),neutrophils (P=2.70×10⁻¹³) and monocytes (P=2.59×10⁻⁷) in the blood wasobserved after 30% transfusion, as compared to pre-transfusion cellcounts. Leukocytes were mobilized, but did not localize to the lung(FIGS. 10A-10B). There was, interestingly, a significant decrease inlymphocytes in the bloodstream after the 30% transfusion (P=0.015) (FIG.10C).

Other animal models of TRALI typically utilize a ‘two-hit’ model thatdirects the inflammatory response to the lungs (reviewed in references).One method of inducing the ‘first hit’ is infusion of LPS which afterintravenous infusion will initially encounter the capillary beds of thelungs likely priming them for overt damage by the ‘second hit’. A 2mg/kg LPS IV injection given as a ‘first-hit’ 30 minutes prior to the‘second-hit’ with transfusion of 30% mismatched erythrocytes resulted indramatic lung damage at 4 hours post transfusion with massive neutrophilinfiltration of lung tissue (FIG. 9D). A corresponding change inintravascular leucocyte levels was also observed with significantreduction of WBC (P=9.8×10⁻⁵), neutrophils (P=4.5×10⁻⁴), monocytes(P=0.004) and a further reduction in lymphocytes (P=0.006) in thebloodstream compared to animals receiving the 30% transfusion in theabsence of the LPS ‘first-hit’(FIGS. 10A-10C). The reduction incirculating leukocytes in this model mimics the transient leukopeniareported in TRALI patients. Together, these results demonstrate thatthis transfusion-initiated model with complement activation results insevere neutrophil-mediated lung disease consistent with many aspects ofTRALI.

Given the inflammatory nature of the intravascular hemolysis observed inthis model, increasing the percentage of transfused human RBCs wastested to determine if it would induce a TRALI-like phenotype in thelungs in the absence of the LPS ‘first-hit’. Transfusion of 15-45% humanRBCs did not result in acute lung injury in the absence of an LPS‘first-hit’. When the LPS ‘first-hit’ was added, a TRALI-likepathogenesis was observed by histology and other hallmarks of neutrophilmediated lung injury were detected such as severe neutrophilinfiltration of lung parenchyma and leukopenia. The need for LPS as a‘first-hit’ to induce TRALI in this model is essential and is consistentwith the numerous ‘two-hit’ rat, mouse, sheep and swine models of TRALIreported in the literature.

To establish a ‘two-hit’ TRALI model, amounts of human RBC (15, 30 or45%) were initially transfused into rats to optimize the Wistar ratmodel. For all procedures, rats were sedated with ketamine andacepromazine throughout the course of the experiment with monitoring ofvital signs. Groups of rats received transfusion of human RBCintravascularly through the indwelling jugular catheter. Blood sampleswere collected into EDTA microtainer tubes (Becton Dickinson) from theanimals prior to transfusion and then at 0.5, 5, 20, 60, 120 and 360 minafter transfusion. These samples were centrifuged at 2,655×g for 5 minto separate out the plasma and sediment the cells. Plasma was aliquotedand the cell pellet was processed separately as described below. Basedon pilot experiments with varying amounts of human RBCs (15-45%), 30%human RBC transfusion produced robust complement-mediated hemolysis over3 hours and was chosen for the TRALI model (FIGS. 8A and 8B).

For the ‘two-hit’ model, rats were sedated as above andlipopolysaccharide (LPS, from Salmonella enterica serotype enteritidis,2 mg/kg [Millipore-Sigma]) was administered intravascularly through theindwelling jugular catheter as the ‘first-hit’ similar to other TRALImodels (reviewed in references). This was followed 30 minutes later by30% mismatched RBC transfusion as the ‘second-hit’. Blood samples werecollected prior to LPS administration and at 4 hours after RBCtransfusion for analysis of blood chemistries (SuperChem and CBC, AntechDiagnostics). Upon completion of the final blood draw, the animals wereeuthanized using isofluorene and guillotine. A necropsy was completed tocollect organs for histopathology. Lungs acquired from each animal wereweighed and then stored in formalin or frozen at −70° C. Hematoxylin andeosin (H&E) stained sections of formalin fixed tissue were reviewed in ablinded fashion.

To determine if prophylactic administration of PIC1 would attenuateTRALI pathogenesis, this transfusion-based antibody-initiatedcomplement-mediated TRALI model was utilized. Twenty-eight minutes afterIV LPS infusion, 160 mg/kg PIC1 or vehicle control was administered IVfollowed by the 30% mismatched RBC transfusion. For animals receivingPIC1, 160 mg/kg of the pegylated derivative, PA-dPEG24 (PolyPeptideGroup) in approximately 0.05 M histidine [pH 6.0], 0.15 M NaCl wasadministered 2 minutes before human RBC transfusion. The selection ofthe 160 mg/kg PIC1 dose was established in the intravascular hemolysisWistar rat model as previously reported. A group of animals receivingvehicle alone and sham animals were also included.

Four hours after transfusion, blood was collected and lung tissueharvested. PIC1 was found to reduce acute lung injury as assessed byhistology and lung weights, neutrophil infiltration of lung tissue,leukocyte sequestration, as well as neutrophil activation as quantifiedby MPO activity and free DNA in the bloodstream. In addition to theTRALI-like injury induced in these animals in the absence of PIC1,significant acute kidney injury (AKI) was observed as assessed by grossmorphology, organ weights and increased blood levels of creatinine,BUN/creatinine and the liver enzyme AST (SGOT) (data not shown) which isconsistent with the AKI previously reported in the AIHTR model that isattributable to free hemoglobin released by hemolysis of the 15%transfused human RBCs. This type of end-organ damage to the kidney hasalso been reported in a mouse model of antibody-mediated TRALI. Whencompared to animals receiving vehicle, PIC1-treated animals had reducedkidney weights and reduction in blood levels of creatinine,BUN/creatinine and AST (data not shown). Thus, even at a 30% transfusionin the presence of LPS pre-treatment, PIC1 protects the kidneys frominjury consistent with the protective effect of PIC1 in the AIHTR model.

Example 3

This Example shows that prophylactic administration of PIC1 reducesacute lung injury. IV administration of PIC1 either immediately beforeor after transfusion of the human RBCs mitigated both hemolysis andkidney damage in an AIHTR model. To ascertain if PIC1 could attenuateTRALI in a novel ‘two-hit’ model, rats were administered 2 mg/kg LPS IVas the first hit. Twenty-eight minutes later, rats received either 160mg/kg PIC1 or vehicle only, followed 2 minutes after that by 30% IVtransfusion of mismatched RBCs as the second hit. The 160 mg/kg dose ofPIC1 was utilized based on its efficacy in the AIHTR model. Four hoursafter transfusion, animals were sacrificed, lung weights weredetermined, and lung tissue evaluated by histology.

Lungs from vehicle control animals exhibited a significant increase inweight compared to sham animals (p=0.006) consistent with increasedcellularity in the lungs (FIG. 11A). In contrast, lungs from thePIC1-treated group showed a significant reduction in lung weightcompared to vehicle control animals (p=0.001) and were not significantlydifferent to that of sham animals (p=0.432) (FIG. 11A). Hematoxylin andeosin (H&E) stained sections revealed that compared to the sham animalswhich demonstrated normal pulmonary histology (FIG. 11B),vehicle-treated animals showed marked consolidation of the alveolarspaces and thickening of the alveolar cell walls with heavy neutrophilinfiltration as expected (FIG. 11C). In contrast, animalsprophylactically treated with PIC1 showed decreased injury to the lungarchitecture (FIG. 11D).

Example 4

This Example shows that PIC1 reduces neutrophil-mediated lung injury,myeloperoxidase (MPO) activity and leukopenia. To evaluate the role ofPIC1 in attenuating neutrophil mediated lung injury in this model, ablinded grading of H&E sections for neutrophil infiltration and cellwall thickening from vehicle and PIC1-treated animals was performed.Neutrophil infiltration and cell wall thickening were scored on a scaleof 0-4 with a score of 0 indicating normal lungs and a score of 4denoting severe lung injury. Consistent with a reduction in lung weightsand histological analysis, animals receiving PIC1 had significantlyimproved lung injury scores compared to vehicle control animals(p=0.003) (FIG. 12A). Vehicle control animals displayed a higher degreeof damage with a mean of 2.0 and a wider distribution compared toPIC1-treated animals with a mean of 1.5 and a much tighter distributionat the 25 and 75 quartiles.

To further evaluate the pathogenic role of neutrophils in this TRALImodel, the effect of PIC1 on neutrophil-mediated lung damage viamyeloperoxidase (MPO) was evaluated. MPO is the major peroxidase enzymein neutrophil granules and contributes to inflammatory lung damage viaformation of hypochlorous acid and other reactive oxygen species as wellas neutrophil extracellular traps (NETs) in many pulmonary diseases. Toascertain if MPO activity could be detected in TRALI lung tissue,extracellular MPO from tissue homogenates of animals receiving PIC1 orvehicle control animals was isolated by antibody capture methodologyfollowed by MPO-mediated oxidation of Amplex Red to fluorescentResorufin in the presence of hydrogen peroxide. Fluorescence was readfrom 0 to 10 minutes every 25 seconds. Compared to animals receivingvehicle, PIC1-treated animals had significantly reduced levels of MPOactivity at 5 (p=0.005) and 10 minutes (p=0.002) (FIG. 12B).

Transient leukopenia may result from neutrophil sequestration in lungtissue and has been observed in animal models of TRALI as well as inpatients with TRALI. See also the data in FIG. 10. To ascertain thecirculating levels of immune cells in vehicle versus PIC1-treatedanimals, blood was recovered prior to euthanasia. Compared to animalsreceiving vehicle, PIC1-treated animals showed a significant increase inblood levels of white blood cells (p=0.043) and lymphocytes (p=0.048)(FIGS. 13A and 13B). Additionally, circulating neutrophils and monocytesalso demonstrated trends towards increased numbers in the bloodstream ofPIC1-treated animals compared to animals receiving vehicle, but did notreach statistical significance (neutrophils, p=0.101; monocytes,p=0.229), (FIGS. 13C and 13D, respectively).

Example 5

This Example shows that PIC1 reduces levels of neutrophil extracellulartrap (NET) biomarkers in circulation. In murine models of TRALI,activated neutrophils can release NETs contributing to acute lung injuryand that the NET biomarker free DNA is elevated in the blood of TRALIpatients. To ascertain whether PIC1 had an effect on the level of freeDNA in circulation, DNA levels in plasma from animals receiving PIC1 orvehicle were quantified in a PicoGreen assay. Compared to animalsreceiving vehicle, the level of free DNA in plasma isolated fromPIC1-treated animals was significantly reduced (p=0.02) (FIG. 14). Takentogether, the reduction of neutrophil-mediated lung injury as assessedby histology, the reduction in MPO activity in the lung tissue, the lackof leukopenia and reduction in free DNA in circulation demonstrate thatPIC1 reduces immune cell-mediated acute lung injury in this novel TRALIanimal model.

Example 6

This example describes experiments to test the extent to which PA-dPEG24could inhibit MPO activity and NET formation in vivo. An inflammatoryperitonitis model in rats was developed utilizing purified MPO injectedintraperitoneally (IP).

Human neutrophil MPO (Lee Biosolutions) was diluted in sterile saline tothe respective doses necessary for each experiment. PIC1 in the form ofPA-dPEG24 (PolyPeptide Group) was solubilized and diluted in 0.5 MHistidine buffer, adjusted to pH 6.5, and then diluted to each of therespective doses used for each experiment.

Wistar rats were sedated and injected IP with purified MPO inexperiments that were conducted under an Eastern Virginia Medical SchoolIRB approved protocol (#17-008). 16-week-old male Wistar rats were usedin these experiments weighing approximately 200 g. The rats receivedsedation with ketamine and acepromazine prior to all procedures. Theythen received a 1 ml IP injection with MPO in saline. After the timeinterval indicated, a tail vein phlebotomy was performed to obtain 250mcl of blood in a K₂EDTA Microtainer (BD). The animals were theneuthanized with FatalPlus IV. A peritoneal wash with 20 ml of ice coldPBS was then performed by IV injection followed by massage andextraction from a small hole in the peritoneum with a blunt needle andsyringe.

Cells were sedimented and supernatants were tested for MPO peroxidationof TMB, with sample processing performed as follows. Peritoneal washfluid was centrifuged at 1500×g for 5 minutes to sediment cells andother debris. The resulting peritoneal wash fluid supernatant wasaliquoted and frozen for further analysis. The cell pellet wasresuspended to the original volume with saline and cells were counted ona hemocytometer. Plasma was collected after centrifugation of thelavender top blood collection tubes.

The MPO activity assay was performed as follows. Peritoneal fluid, alongwith pure MPO for a standard curve, was serially titrated in 0.1 ml in a96 well plate followed by the addition of 0.1 ml of3,3′,5,5′-tetramethylbenzidine (TMB) (Fisher). After one minute, 0.1 mlof 2.5 M H₂SO₄ was added to stop the reaction and the plate was read at450 nm. Using linear regression from the standard curve, the MPOactivity was calculated for each sample.

MPO-mediated peritonitis dose-response experiments were performed asfollows. A dose response pilot experiment was performed to determine adose of purified MPO that would demonstrate MPO-mediated peroxidaseactivity and NET formation. Purified MPO was injected IP into the ratsat doses of 0.01, 0.03 and 0.1 mg. After one hour, the rats wereeuthanized, and peritoneal lavage was performed. The results are shownin FIG. 17. With the MPO dose increasing to 0.1 mg, a trend towardsincreased peroxidase activity (P=0.12) in the peritoneal lavage fluidmeasured by a TMB-based assay was demonstrated.

In the peritoneal wash fluid supernatants, free DNA was measured as amarker of NETosis via the PicoGreen assay. Peritoneal fluid or plasma,along with DNA (Invitrogen) for a standard curve, was serially titratedin 0.1 ml in a 96 well plate and used in the Quant-iT PicoGreen (Fisher)assay. The PicoGreen solution was diluted as directed by the kitinstructions and added to samples, which were incubated in the dark for10 minutes and then read on a fluorescent microplate reader atexcitation 480 nm/emission 520 nm). Using linear regression from thestandard curve, the DNA concentration was quantitated for each sample.

The results shown in FIG. 18. A trend towards increased free DNA wasnoted as the dose increased to 0.1 mg MPO dose (P=0.19). The results ofthese experiments suggested a dose-response relationship where thehighest dose of MPO, i.e., 0.1 mg, was more likely to show more activitythan the lower doses tested.

To summarize, MPO injected IP in rats resulted in increased peroxidationactivity in the peritoneum (FIG. 17) and increased free DNA that issuggestive of NETosis (FIG. 18).

MPO-mediated peritonitis time course experiments were then performed. Atime course experiment was conducted to evaluate the optimal dwell timefor the purified MPO in the peritoneum in this model. MPO wasadministered IP at a higher 0.1 mg dose and peritoneal washes wereperformed after euthanasia at 1, 2 and 4 hours after MPO injection. Asshown in FIG. 19, a ten-fold increase in MPO peroxidase activity wasdemonstrated at 2 hours after injection (P=0.005) without furtherincrease at 4 hours. An eight-fold increase in free DNA was demonstratedat 2 hours after injection (P=0.03), as shown in FIG. 20. These resultssuggested that purified MPO injected IP increased peroxidase activityand NETosis in the peritoneal wash fluid at 2 hours after injection.

The effect of PIC1, in particular PA-dPEG24, on the inhibition of MPOand NETosis in MPO-mediated peritonitis was then assayed. A series ofincreasing doses of PA-dPEG24 (1 mg, 5 mg, and 20 mg) were injected IPimmediately, i.e., one minute after purified MPO was inoculated, intothe peritoneum followed 2 hours later by phlebotomy, euthanasia andperitoneal wash. A control was used where no PA-dPEG24 was administered.An MPO activity assay and a free DNA assay according to the methodsdiscussed above were conducted for each of 0 mg, 1 mg, 5 mg, and 20 mgPA-dPEG24 doses.

The results for the MPO activity assay are shown in FIG. 21 and thosefor the free DNA assay (in peritoneal wash supernatant free DNA) areshown in FIG. 22. PIC1 injected IP in this model showed a decreasedperoxidation activity in the peritoneum (FIG. 21) and showed a trendtowards decreased NETosis (FIG. 22). In particular, a 20 mg dose (100mg/kg) of PA-dPEG24 demonstrated a 5-fold decrease (P=0.015) inperoxidase activity by TMB compared with no PA-dPEG24 after MPOinjection. Statistically significant decreases in MPO activity (P<0.019)were also demonstrated for PA-dPEG24 doses of 5 mg (25 mg/kg) and 1 mg(5 mg/kg). A trend towards decreased free DNA in the peritoneal fluid(P=0.11) was noted for the 20 mg (100 mg/kg) dose of PA-dPEG24, ascompared with no PA-dPEG24.

Free DNA was also measured in the blood plasma, with the results shownin FIG. 23. A similar trend was found in which free DNA was lowest withthe highest dose of PA-dPEG24 (P=0.22), as compared with no PIC1 afterMPO injection. These results demonstrate that PA-dPEG24 can decreaseMPO-mediated peroxidase activity in vivo in this purified MPOperitonitis model. The results also suggest that PA-dPEG24 may be ableto decrease MPO-mediated NETosis in vivo.

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All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

What is claimed is:
 1. A method of treating systemic lupus erythematosus(SLE) in a subject comprising administering a therapeutically effectiveamount of PIC1 to the subject.
 2. The method of claim 1, wherein themethod is effective to modulate immune complex activation of thecomplement system and NET formation in the subject.
 3. The method ofclaim 2, wherein the NET formation is stimulated by at least one of abacterium, a fungus, a parasite or a virus.
 4. The method of claim 1,wherein the method is effective to inhibit NET-mediated inflammatorytissue damage in the subject.
 5. A method of treatingtransfusion-related acute lung injury (TRALI) in a subject comprisingadministering a therapeutically effective amount of PIC1 to the subject.6. The method of claim 5, wherein the method is effective to modulateimmune complex activation of the complement system and NET formation inthe subject.
 7. The method of claim 6, wherein the NET formation isstimulated by at least one of a bacterium, a fungus, a parasite or avirus.
 8. The method of claim 5, wherein the method is effective toinhibit NET-mediated inflammatory tissue damage in the subject.
 9. Themethod of claim 5, wherein the PIC1 is administered before the subjectis administered a blood transfusion, after the subject is administeredthe blood transfusion, and/or during the blood transfusion.
 10. Themethod of claim 1, wherein the PIC1 inhibits myeloperoxidase (MPO)activity in the subject.
 11. The method of claim 1, wherein the PIC1 isadministered parenterally.
 12. The method of claim 1, wherein thesubject is human.
 13. The method of claim 4, wherein the subject ishuman.
 14. The method of claim 5, wherein the subject is human.
 15. Themethod of claim 1, wherein the PIC1 is a peptide comprising one or morePEG moieties.
 16. The method of claim 15, wherein the PIC1 is PA-dPEG24.17. The method of claim 16, wherein the PA-dPEG24 comprises the sequenceof IALILEPICCQERAA-dPEG24 (SEQ ID NO: 19).